Apparatus for detecting particles

ABSTRACT

Apparatus for detecting particles in a liquid, comprises an evanescent optical sensor having an electrically conducting layer, and means associated therewith for providing an electric field at or adjacent the sensor surface. Preferably, the optical sensor is a surface bound sensor based on a metal-clad leaky waveguide structure. A DC potential applied to the metal layer and a parallel counter electrode provides a uniform electric field, which drives deposition of the particles on the sensor surface. Alternatively, an alternating potential applied to a electrically conducting layer configured as an electrode and counter electrode induces an electro-osmotic bulk flow in the liquid which drives deposition of the particles on the sensor surface.

The present invention is generally concerned with apparatus for enhanceddetection of particles using optical sensors. The invention isparticularly, but not exclusively, directed towards enhanced depositionof particles, such as bacteria and viruses, on surface bound opticalsensors.

As used herein the term “surface bound sensor” describes an opticalsensor including a sensing medium comprising a biological materialcapable of binding the particles to be detected—such as an antibody orlectin.

Optical sensors often comprise waveguide structures in which anevanescent wave, associated with an optical mode existing in thestructure, extends into the sensing layer. A change in the refractiveindex of the sample by interaction with a particle leads to a change inan optical property of the mode, which can be readily detected.

One such optical sensor uses the phenomenon of surface plasmon resonance(SPR). Light incident a dielectric prism having an upper surface coatedwith a thin metal layer of gold or silver and a sensing layer comprisingthe biological sample, is coupled at a certain “resonant” angle orangles to oscillations of the electron cloud in the metal layer. Asurface optical mode is propagated at the interface of the prism andmetal layer and a drop in the amount of reflected light is recorded at adetector. The surface optical mode generates an evanescent field thatextends into the sensing medium. A particle binding to the sample leadsto a change in the refractive index of the medium affecting the surfacemode and the angle at which resonance is excited.

The basic structure of “leaky” waveguide sensors, described inInternational Patent Application WO 99/44042, is similar but offersimproved sensitivity in that an optical mode is supported in the bulk ofthe sensing layer.

International patent application WO 01/42768A describes flow cellapparatus in which detection of surface bound particles on an SPR sensoris improved by monitoring light scattered or emitted there from.

Improved detection is also obtained using metal-clad or dye-clad leakywaveguide sensors described in our co-pending international patentapplication PCT/GB02/045045 incorporated by reference herein. Here, anevanescent wave extends above the sensing surface and can be “tailored”,by choice of refractive indices in the stucture, to optimise overlapwith the particles to be detected.

However, the sensitivity of flow cell apparatus incorporating theseoptical sensors is inherently limited by slow diffusion of the particlesin the liquid to the sensor surface, even with stirring or agitation.Consequently, there is still a need to improve the detection ofparticles at surface bound sensors.

The present invention generally aims to improve detection at opticalsensors, and in particular, surface bound optical sensors by providingan electric field acting on the particles so as to direct them to thesensor surface.

Under suitable fluid flow conditions, the electric field may act oncharged particles or on an induced dipole on the particles to enhancethe deposition of the particles to the sensor surface.

Accordingly, in one aspect, the present invention provides apparatus fordetecting particles in a liquid, comprising an evanescent opticalsensor, including an electrically conducting layer, and means associatedtherewith for providing an electric field at or adjacent the sensorsurface.

It will be apparent, that the electrically conducting layer can initself comprise an electrode. In a preferred embodiment of the presentinvention, therefore, the means providing an electric field comprise aplane surface counter electrode arranged in parallel with the conductinglayer, a DC potential source and means for applying the potential acrossthe electrode and the conducting layer. In this embodiment, theconducting layer and the plane surface electrode together define a gapelectrode configuration in which the sensing layer partially extends.

It will be understood that the DC potential provides a uniform electricfield capable of charging the particles and directing them to the sensorsurface (electrophoresis). The magnitude of the DC potential, the extentof the gap between the plane surface electrode and the conducting layer,the flow rate of the liquid containing the particles across the sensorsurface will all be determined having regard to each other and to theparticle size. It will be appreciated that the force acting on theparticles should be sufficient to overcome flow and drag effects.

In a preferred embodiment of the present invention, the optical sensorcomprises a surface bound sensor such as the MCLW sensor mentioned aboveand described in our co-pending international patent applicationPCT/GB02/045045. Suitable electrically conducting layers comprisemetals, in particular, aluminium, tantalum, zirconium, titanium orchromium, or crystalline dye materials.

Suitable applied potentials for the detection of bacillus subtilis var.Niger (bacillus globbiggi, BG) spores according to this embodiment rangefrom 10 to 100 V, typically about 30 V for a gap size ranging from 20-50μm and flow rates ranging from 50 to 300 μl min⁻¹.

The plane surface counter electrode may comprise a conducting, metal ormetal oxide layer arranged on an inert substrate. Preferably, thecounter electrode comprises a layer of indium tin oxide (ITO) arrangedon a glass substrate since it is clear and known to resist opticaldegradation on prolonged polarisation by application of an alternatingpotential.

In another embodiment of the present invention, the electricallyconducting layer of the optical sensor is itself configured, asdescribed in our co-pending GB patent application No.0303305.7—incorporated by reference herein, so as to provide a planarelectrode and planar counter electrode.

In this embodiment, the means providing an electric field at or adjacentthe sensor surface comprise an alternating potential. The alternatingpotential when applied at a predetermined frequency and magnitude to thelayer induces a non-uniform electric field that can induce anelectro-osmotic flow in the bulk of the liquid so as to focus particlesonto the sensor surface (abnormal dielectrophoresis).

It will be understood that, in some embodiments of the presentinvention, the fluid layer in contact with the upper surface of thesensor comprises a sensing layer. The fluid layer, which issemi-infinite, will contain the particles to be detected. However, asurface bound sensor is preferred, particularly where it comprises asensing layer of an antibody or lectin.

In a second aspect, the present invention provides a method fordetecting particles in a liquid comprising i) introducing the liquid toan evanescent optical sensor, including an electrically conductinglayer, having means associated therewith for providing an electric fieldat or adjacent the sensor surface and ii) generating the electric field.

In a preferred embodiment, the means providing an electric fieldcomprise a plane surface counter electrode arranged in parallel with theconducting layer, a DC potential source and means for applying thepotential across the electrode and the conducting layer. In thisembodiment, the conducting layer and the plane surface electrodetogether define a gap electrode configuration in which the sensing layerpartially extends.

The magnitude of the DC potential, the extent of the gap and the flowrate of the liquid containing the particles across the sensor surfacewill all be determined having regard to each other and to the forcenecessary for the particles to overcome flow and drag effects.

In a particularly preferred embodiment, the optical sensor comprises asurface bound optical sensor such as the MCLW sensor described in ourco-pending international patent application PCT/GB02/045045.

The DC voltage may be applied to one or both of the electrodes.Preferably, however, the counter electrode is earthed. Suitable appliedpotentials for the detection of bacillus subtilis var. Niger (bacillusglobbiggi BG) spores according to this embodiment from 10 to 100 V,typically about 30 V for a gap size ranging from 20-50 μm and flow ratesranging from 50 to 300 μl min⁻¹.

Other embodiments in this aspect of the present invention will beapparent from the foregoing description. In addition, it will beapparent that a combination of uniform and non-uniform fields(electrophoresis and dielectrophoresis) maybe used where it is desiredto manipulate different particles.

The present invention will now be described by reference to severalembodiments and the following examples and drawings in which

FIG. 1 is a schematic illustration of one embodiment of the presentinvention;

FIGS. 2 a) to c) are photographs showing electrophoretic enhancement forthe collection of bacillus globiggi BG spores on the embodiment of FIG.1;

FIG. 3 is a graph showing the enhancement at various concentrations ofspores;

FIG. 4 is a graph showing the capture of spores on a preferredembodiment of the present invention; and

FIGS. 5 a) and b) show the scattering image and fluorescence image ofthe spores captured on the preferred embodiment.

Having regard now to FIG. 1, a basic MCLW chip 11 comprises an uppersurface of a 300 nm silica sol layer 12 (n=1.43) provided on a thinlayer 13 (8.5 nm) of titanium coating a 1 mm glass substrate layer 14(n=1.5). The thickness and refractive index of the silica sol layer 12is chosen to support a single sharp-guided optical mode at a wavelengthof incident light of 685 nm or 488 nm and to optimise the extent of theevanescent field above the surface to about 1.5 to 2.0 μm.

As mentioned previously, a final sensing layer (not shown) may comprisea liquid layer containing the particles to be analysed. Alternatively oradditionally the sensing layer can comprise an antibody layer depositedon the silica sol layer.

A counter electrode comprises a glass substrate 15 coated with aconducting layer 16 of ITO. The counter electrode is joined to the chip11 by a 30 μm (gap size) double-sided adhesive tape 17 through which a12 mm by 2 mm section, defining a flow cell 18, has been cut. Two 1.5 mmholes drilled in the counter electrode provide inlet and outlet meansfor the flow of liquid through a delivery and collection tube 19 to thecell 18.

The titanium layer 13 of the sensor chip is provided with a silverloaded epoxy contact (not shown) at one end of the chip 11. The ITOlayer 16 of the counter electrode is provided with similar contacts (notshown) at positions along its length so as to reduce voltage gradientdue to the resistance of the metal and ITO layers. The contacts connectthe sensor chip and counter electrode to a DC potential source.

The assembly is used in conjunction with an equilateral, coupling prism(not shown 30 mm) of BK 7 glass and refractive index 1.510. Theinterrogation of particle deposition at the upper surface of the chipmay be conducted using the basic arrangement including an optical sourceand detection means described in our co-pending international patentapplication PCT/GB02/045045.

EXAMPLE 1

The sensor surface was blocked by exposure to 0.1% w/v BSA in PBS/Tween®20 and stored overnight at 4° C. BG spores (4.7×10⁷ spores/ml) in 50 mMhistidine buffer were introduced to the flow cell using a MINIPULS-3,MP4 peristaltic pump (Gilson, Canada) at 50 μl min⁻¹. A positivepotential of 30 V was applied to the metal sensor layer with respect tothe counter electrode for a total of 2 min. After a further period, anegative potential of 1.5 V was applied to the metal sensor layer withrespect to the counter electrode.

FIG. 2 shows the distribution of spores before (a) and on (b) applyingthe positive potential. As may be seen, the number of cells incident thesensor surface is greatly increased during the application of thepotential. The application of the negative potential (c) highlightsnegligible non-specific adsorption of the spores to the chip surface.

These effects were repeated for BG spore concentrations ranging from 10³to 10⁶ spores/ml. An increase in the electrical conductivity of thebuffer (to 150 mM NaCl), however, greatly decreased the number of cellsincident the sensor surface—presumably through ionic screening andrestriction of the electric field to the double charge layer adjacentthe electrodes.

EXAMPLE 2

A direct immunoassay utilised two DC potential application steps. Abiotinylated-labelled anti BG capture antibody was deposited on the chipby repeated introduction of 50 μg/ml suspension in water to the flowcell (200 μl/min) and applying a positive potential of 20 V. Unboundantibody was removed by washing with 50 mM histidine buffer. A BG sporesolution (4×10⁷ spores in 50 mM histidine buffer) was introduced to theflow cell at a flow rate of 50 μl/min. A positive potential of 30 V wasapplied to the metal sensor layer with respect to the counter electrodeduring 2 min. After a short period, a negative potential (1.5 V) wasapplied to the metal sensor layer with respect to the counter electrode.The assay was visualised in real time by detection of scattered lightfrom captured BG spores using a CCD camera. These effects were repeatedfor BG spore concentrations ranging from 10³ to 10⁶ spores/ml (FIG. 3).

FIG. 4 shows the number of cells captured by subtraction of the numberof spores removed from the sensor surface on application of the negativepotential from the number of spores. The values are in good agreementwith the number of captured spores remaining on the surface. Comparisonof the number of captured spores with the number of spores capturedusing the MCLW chip alone revealed a 30 fold enhancement equal to anincrease in local concentration from 10³ to 10⁸ spores.

EXAMPLE 3

A sandwich format immunoassay utilised three DC potential applicationsteps. A biotinylated-labelled anti BG capture antibody was deposited onthe chip and BG spores were captured on the antibody layer as previouslydescribed. Finally a suspension of CY5-labelled BG antibody (10 μg ml⁻¹in 50 mM histidine buffer) was introduced to the cell at a flow rate of50 μl min⁻¹ over 2 min. A positive potential of 30 V was applied to themetal sensor layer with respect to the counter electrode during 2 min.Unbound CY5-labelled antibody was washed from the flow cell by purgingwith 50 nM histidine buffer for 3 min at 200 μl min⁻¹.

FIG. 5 shows the scattering image of captured BG spores and thefluorescence image following exposure to CY5 labelled antibody. Althoughnot shown it is found that the fluorescence emitted from the labelledantibody is greater when attached to the captured spores than when thelabelled antibody alone is deposited at the sensor surface.

Repetition of the experiment with varying concentration of labelledantibody (3, 10, 20 μg ml⁻¹) showed that the optimum for detection ofcaptured spores by fluorescence using this method was 10 μg ml⁻¹.

1. Apparatus for detecting particles in a liquid, comprising anevanescent optical sensor having an electrically conducting layer, andmeans associated therewith for providing an electric field at oradjacent the sensor surface.
 2. Apparatus according to claim 1, in whichthe optical sensor comprises a surface plasmon resonance chip. 3.Apparatus according to claim 1, in which the optical sensor comprises ametal-clad leaky waveguide chip.
 4. Apparatus according to claim 3, inwhich the means for providing an electric field comprise a plane surfacecounter electrode and a DC potential source.
 5. Apparatus according toclaim 4, in which the counter electrode is arranged parallel to theconducting layer.
 6. Apparatus according to claim 4, in which thecounter electrode and sensor define a gap there between ranging from 20to 50 μm.
 7. Apparatus according to claim 4, in which the counterelectrode comprises a layer of indium tin oxide provided on a glasssubstrate.
 8. Apparatus according to claim 3, in which the electricallyconducting layer comprises titanium.
 9. Apparatus according to claim 1,in which the optical sensor is a surface bound sensor.
 10. A method fordetecting particles in a liquid comprising i) introducing the liquid toan evanescent optical sensor, including an electrically conductinglayer, having means associated therewith for providing an electric fieldat or adjacent the sensor surface and ii) generating the electric field.11. A method according to claim 10, in which the optical sensorcomprises a surface plasmon resonance chip.
 12. A method according toclaim 10, in which the optical sensor comprises a metal-clad leakywaveguide chip.
 13. A method according to claim 12, in which thegenerated electric field is uniform.
 14. A method according to claim 13,in which a DC potential is applied to a gap electrode configurationcomprised by the sensor and a parallel planar surface counter electrodeat magnitude ranging from 10 to 100 V for a gap size ranging from 20 to50 μm and a flow rate ranging from 50 to 300 μl min⁻¹.
 15. A methodaccording to claim 10, in which the optical sensor is a surface boundsensor.
 16. (canceled)
 17. (canceled)